Project Summary/Abstract Broadly neutralizing antibodies (bnAbs) that arise during chronic HIV infection highlight the remarkable capacity of the human immune system to evolve antibodies that can recognize highly variable and glycosylated proteins. Eliciting these types of antibodies via a prime-boost vaccine however remains a difficult challenge. Recently, structure-based rational vaccine design strategies have reinvigorated the pursuit of an effective HIV vaccine. This approach relies on a high-resolution understanding of the interaction between the sole neutralizing target on the surface of HIV, envelope glycoprotein (Env), and bnAbs that can be used to engineer immunogens designed to recapitulate bnAb responses in nave individuals. This approach is iterative and relies on the ability to characterize immune responses in animals to such immunogens to refine the immunogen design. Serum neutralization assays and ELISA binding provide an estimation of the elicited immune response but not the molecular details required for rational design. Epitope mapping via alanine scanning, antigen specific monoclonal antibody isolation, and next generation sequencing provide further details, but each has limitations and are very time-consuming endeavors. We have therefore developed a novel method to dissect polyclonal immune responses to provide a complete and quantitative map of the epitopes targeted that is rapid and can inform boosting strategies in real time. By purifying immune complexes formed between Env trimer immunogens and serum antibodies from immunized animals, and imaging them by electron microscopy, we directly visualize polyclonal antibody responses. These responses can be quantitated directly or in combination with deep sequencing and mass spectroscopy. We have shown proof of concept in rabbits immunized with BG505 SOSIP.664 native-like Env trimers, and propose to use our methodology to study a recently completed large non-human primate (NHP) vaccination study (138 animals) that tested various immunization strategies using the BG505 SOSIP.664 platform. Within this study there are animals that did or did not develop potent autologous neutralizing antibody responses, as well as animals that developed some level of neutralization breadth. Hence, we have a unique opportunity to understand the molecular underpinnings of such immune responses, and to delineate phenotypic signatures within the polyclonal antibody responses that can be used to guide HIV vaccine development. Further, our studies can be applied to human serum samples for an upcoming human clinical trial using the same BG505 SOSIP.664 immunogen. And thus, we can generate comparative data between humans and NHPs to improve the utility of pre-clinical animal models that remain an essential component of the iterative rational HIV vaccine design process.